Plasmodesmata (PD) are membrane-lined channels that span the plant cell wall, linking the cytoplasm between adjacent plant cells. PD form a continuum of cytoplasmic bridges enabling intercellular transport of solutes, signaling molecules, protein, RNA, and ribonucleoprotein complexes. As PD are essential gatekeepers for cell-to-cell transport during development and for the spread of plant viruses and gene silencing signals, our studies will enhance our understanding of how PD control complex developmental programs and plant defense. As plants are important to human health, our studies will lead to the design of strategies to increase plant defense to plant viruses that pirate PD channels, thereby increasing the productivity of agriculturally important crops. We developed a unique genetic screen that assays for altered PD mediated cell-to-cell transport during Arabidopsis embryonic development. To date we have analyzed two mutants, ise1 and ise2, that cause embryos to increase PD mediated traffic of fluorescently tagged probes. ISE1 and ISE2 encode two distinct types of RNA helicases with independent roles in PD mediated transport. ISE1 localizes to PD and thus will provide direct insight into plant cell-to-cell transport. ISE2 does not localize to PD. However, ise2 mutants contain PDs that are altered in structure, localization and frequency. Thus, we will use ise2 as a means to directly identify PD components that are altered in ise2 compared to wild type. The proposed studies have 3 distinct aims: 1.To characterize ISE1 and ISE2 biochemical properties (helicase activity), determine their gene expression patterns, create transgenic lines for their over- and under-expression, and develop conditions for viral induced gene silencing of their genes. 2. Three types of analyses will characterize ISE1. A. Based on the fact that ISE1 also localizes to microtubules, we will perform in depth analyses of ISE1 microtubule association and PD targeting. B. We will study whether ISE1 exerts a positive or negative role in PD mediated intercellular transport by assaying protein (GFP), viral RNA, and gene silencing RNA movement when ISE1 is increased or decreased. C. We will identify ISE1 interaction partners. 3. As ise2 mutants alter PD architecture, localization, and frequency, we will identify what PD components are altered in ise2 compared to wild type. Identification of altered PD components in ise2 provides a direct means to identify PD structural components. Our lab is uniquely poised to carry out the above experiments, as we have developed all the biological tools, and have accumulated expertise in the proposed technical approaches. PUBLIC HEALTH RELEVANCE Intracellular and intercellular signaling pathways are critical for the coordination of gene expression during growth and development in multicellular organisms. While intracellular signaling components and their pathways are conserved in animals and plants, plants have unique plasmodesmata (PD) intercellular connections. As PD are essential gatekeepers for cell-to-cell transport during plant development, these studies will impact how transport of RNA and protein controls complex developmental programs, and also have application to plant defense mechanisms (as PD function to limit virus spread and transport of gene silencing signals).